*3.1. Chemicals*

All chemicals were of analytical reagen<sup>t</sup> grade and used as received. HPLC-grade acetonitrile and methanol were purchased from Labscan (Dublin, Ireland). Acetic acid of analytical grade (assay >99.5%) was purchased from Fluka (Buchs, Switzerland). Double-deionized water with a conductivity of <18.2 MΩ was obtained with a Milli-Q system (Millipore, Bedford, MA). Standard compounds such as hydroxytyrosol, vanillin, luteolin, apigenin, ferulic acid, oleanolic, and maslinic acids were purchased from Sigma–Aldrich (St. Louis, MO, USA,) (+)-pinoresinol was acquired from Arbo Nova (Turku, Finland), and oleuropein from Extrasynthèse (Lyon, France).

### *3.2. Sampling and Drying*

Olive leaves were collected from the olive cultivar 'El Hor' from the Center of Tunisia. The collection of the leaf samples began from January 2017 and continued until November 2017. The collected olive leaves were directly transferred to the laboratory, washed with distilled water. Portions of leaves were immediately stored at −80 ◦C and other portions were dried either at room temperature (25 ◦C) in a programmable mechanical convection oven (Binder Gmbh) or various temperatures of 40, 60, 80, 100, and 120 ◦C. Leaves were ground using an Ultra Centrifugal Mill ZM 200 (Retsch Gmbh, Germany). As a means of gaining representative results and eliminating factors which could affect the monitoring of the phenolic compounds, this fraction was isolated by supercritical CO2 extraction without storage of dried samples.

### *3.3. Supercritical CO2 Extraction*

Supercritical CO2 extraction was carried out with a Waters Prep Supercritical Fluid Extraction system (SFE-100) equipped with a high-pressure CO2 P-50 pump, a high-pressure co-solvent P-50 pump, an automated back pressure regulator, a low-pressure heating exchange, a high-pressure heating exchange, a high-pressure extraction, and a high-pressure collection vessel. Prior to the extraction process, 10 g of ground olive leaves were homogenized with 15 g of sea sand, that was selected as inert material to hold the sample inside the extraction cell and to improve extraction efficiency. This mixture was introduced into the extraction cell and packed with glass wool. Extractions were carried out at 150 bar and 40 ◦C, once experimental conditions had been reached, the extraction solvent (consisting of a mixture of CO2 plus 6.6% of ethanol as a modifier) passed through the extraction cell for one hour at 23 g/min. The obtained extract was collected, and the solvent was evaporated under vacuum at 38 ◦C [9]. A minimum of three replicate extractions was performed for each plant sample, and each extract was analyzed at least three times by HPLC analysis.

### *3.4. RP-HPLC-TOF MS Analysis of Phenolic and Terpenoid Compounds*

Analytical methods to characterize the phenolic and terpenoid compounds in olive leaf SFE extracts were performed in an Agilent 1200 series Rapid Resolution LC (Agilent Technologies, CA, USA) equipped with a vacuum degasser, autosampler, a binary pump, and a diode array detector (DAD). The HPLC system was coupled to a micrOTOF (BrukerDaltonics, Bremen, Germany), and an orthogonal-accelerated TOF mass spectrometer using an electrospray interface (model G1607A from Agilent Technologies, Palo Alto, CA, USA). The chromatographic separation of these compounds was carried out on a C18 Zorbax Eclipse Plus analytical column (4.6 × 150 mm, 1.8 μm) from Agilent Technologies.

Regarding phenolic compounds, the mobile phases used were water with acetic acid (0.5%) (mobile phase A), and acetonitrile (mobile phase B) and the solvent gradient changed according to the following conditions (Table 2):


**Table 2.** Solvent gradient conditions for the analysis of phenolic compounds.

The injection volume in the HPLC was 10 μL. The flow rate used was set at 0.80 mL/min throughout the gradient and, consequently, the use of a splitter was required for the coupling with the MS detector, as the flow that arrived at the TOF detector had to be 0.2 mL/min in order to obtain reproducible results and stable spray. Source and transfer parameters for MS analysis were set using the negative ion mode with spectra acquired over a mass range from *m/z* 50 to 1000 [9]. External mass spectrometer calibration was performed with sodium acetate clusters (5 mM sodium hydroxide in water/2-propanol 1/1 (*v/v*), with 0.2% of acetic acid) in quadratic high-precision calibration (HPC) regression mode. The calibration solution was injected at the beginning of the run, and all the spectra were calibrated prior to polyphenols identification.

Separation of triterpenic compounds from olive leaf extracts was performed on the same 1200 series Rapid Resolution LC (Agilent Technologies, CA, USA) described above. The mobile phases used were acidified water with 0.1% formic acid as mobile phase A and methanol with 0.1% formic acid as mobile phase B (Table 3). The gradient was as follows:


**Table 3.** Solvent gradient conditions for the analysis of triterpenoids.

The injection volume in the HPLC was 10 μL. The flow rate was at 0.8 mL/min. The same MS conditions as described above were used, using sodium formate clusters for external mass spectrometer calibration.

Quantitation was carried out by HPLC-ESI-TOF-MS. Nine standard calibration curves of the main compounds found in samples were prepared using nine commercial standards. The stock solutions containing these analytes were prepared in methanol/water (50/50, *v/v*). All calibration curves showed good linearity over the range of study with a minimum value of R<sup>2</sup> = 0.992. Results are given in mg of analyte per g of olive leaf for triterpenoids and in μg of analyte per g of olive leaf for the rest of compounds.
